Umbilical cord mesenchymal stem cell‐derived exosomes promote axon regeneration during optic nerve injury through microRNA‐dependent mTORC1 signalling

Dear Editor, Treatment for optic nerve (ON) injury remains a challenge worldwide. As essential paracrine signals of mesenchymal stem cells (MSCs), exosomes (exos) show source cell-like biological functions, which have recently been suggested to promote the functional recovery of central neurons.1–4 Reportedly, MSC-exos transfer active components, especially microRNAs (miRNAs), to facilitate intercellular communication, thereby exerting tissue regenerative effects.5–8 However, the effect of exos derived from umbilical cord MSCs (HuMSC-exos) and the underlying mechanisms in ON injury remain unclear. Here, we intended to explore whether HuMSC-exos have a neuroregenerative effect and the molecular mechanisms by which exosomal miRNAs play a role in this process. Exos were isolated from HuMSCs (Figure S1A–C) and purified by ultracentrifugation. Purified HuMSC-exos were identified by positive expression of their specific surface markers (CD9, CD63 and TSG101) by Western blotting (Figure S2A–E). They displayed typical exosomal features, including morphology and size, as detected by transmission electron microscopy (Figure S2F). Nanoparticle tracking analysis revealed that purified HuMSC-exos were relatively homogeneous particles, and the particle concentration in the distribution range of 30–200 nm was approximately 93.3% (Figure S2G). In the ON crush (ONC) model, HuMSC-exos promoted the survival of 30% of the retinal ganglion cells (RGCs) (Figure 1A,B), rescued 60% of the RGCs from apoptosis (Figure 1C,D), increased retinal nerve fibre layer thickness (Figure 1E–G) and promoted axon regeneration (Figure 1H–J) at 21 days after ONC. In addition, pattern electroretinography showed thatHuMSC-exos efficiently recovered the loss of RGC function after ONC (Figure 2A–C). Intravitreal injection ofHuMSC-exos rarely caused retinal detachment, and no other side effects were

Dear Editor, Treatment for optic nerve (ON) injury remains a challenge worldwide. As essential paracrine signals of mesenchymal stem cells (MSCs), exosomes (exos) show source cell-like biological functions, which have recently been suggested to promote the functional recovery of central neurons. [1][2][3][4] Reportedly, MSC-exos transfer active components, especially microRNAs (miRNAs), to facilitate intercellular communication, thereby exerting tissue regenerative effects. [5][6][7][8] However, the effect of exos derived from umbilical cord MSCs (HuMSC-exos) and the underlying mechanisms in ON injury remain unclear. Here, we intended to explore whether HuMSC-exos have a neuroregenerative effect and the molecular mechanisms by which exosomal miRNAs play a role in this process.
Exos were isolated from HuMSCs (Figure S1A-C) and purified by ultracentrifugation. Purified HuMSC-exos were identified by positive expression of their specific surface markers (CD9, CD63 and TSG101) by Western blotting ( Figure S2A-E). They displayed typical exosomal features, including morphology and size, as detected by transmission electron microscopy ( Figure S2F). Nanoparticle tracking analysis revealed that purified HuMSC-exos were relatively homogeneous particles, and the particle concentration in the distribution range of 30-200 nm was approximately 93.3% ( Figure S2G).
In the ON crush (ONC) model, HuMSC-exos promoted the survival of 30% of the retinal ganglion cells (RGCs) ( Figure 1A,B), rescued 60% of the RGCs from apoptosis ( Figure 1C,D), increased retinal nerve fibre layer thickness ( Figure 1E-G) and promoted axon regeneration ( Figure 1H-J) at 21 days after ONC. In addition, pattern electroretinography showed that HuMSC-exos efficiently recovered the loss of RGC function after ONC (Figure 2A observed in the ONC model. Therefore, intraocular injection is safe. Consistently, we also observed that 3 × 10 9 HuMSC-exos promoted RGC neuritogenesis in retinal cell culture ( Figure S3) and were more effective than 3 × 10 9 BMSC-exos ( Figure 2D-G). In contrast, the higher doses of HuMSC-exos significantly reduced the number of RGCs with neurites compared with that in the untreated cell culture ( Figure 2D,F). Together, these results suggest that a moderate dose of HuMSC-exos has a neuroregenerative effect after ON injury.
An increasing number of studies show that exos as nanocarriers are important mediators that facilitate intercellular communication rather than cell-to-cell contact. Previous reports have shown that exo-mediated miRNA delivery is a new method of intracellular communication and plays a key role in central nervous system (CNS) therapy. [5][6][7] However, studies on the role of HuMSC-exos and exosomal miRNAs in ON injury are very rare. Thus, using high-throughput sRNA sequencing, we analyzed miRNA profiles in HuMSC-exos. The data showed that 24 specific miRNAs were abundant in HuMSC-exos compared with HEK293T-exos ( Figure 3A and File S1). KEGG pathway analysis showed that the target genes of these twenty-four miRNAs were significantly enriched in the mTOR pathway ( Figure S4A and File S2). GO functional annotation analysis revealed that the target genes associated with axonogenesis, axon extension and neurogenesis were significantly enriched ( Figure S4B and File S3). Next, we verified that eight of 24 miRNAs could be delivered into RGCs by HuMSC-exos, miR-22-3p, miR-222-3p, miR-221-3p, miR-21-5p, miR-543, miR-29a-3p, miR-24-3p and miR-27a-3p, which accounted for 17.79% in HuMSC-exos but only 6.65% in HEK293T-exos ( Figure 3B,C). Then, we used TargetScan, miRDB, miRWalk and miRTarBase to identify that these miRNAs all directly target mTOR pathway-related genes ( Figure S4C and File S4). mTOR acts throughtwo signalling complexes called mTORC1 and mTORC2. Activation of mTORC1 leads to     the downstream phosphorylation of S6 ribosomal protein (pS6) to initiate protein translation. 9 Recent studies have shown that an increase in mTORC1 activity may be an important factor for regeneration in the mature CNS. 10 Therefore, the determination of whether HuMSC-exos play a regenerative role by regulating mTORC1 activity in ON injury is very important. Immunostaining of pS6 was used to monitor mTORC1 activity, and the results indicated that HuMSC-exos significantly increased the mTORC1 activity of RGCs both in vitro and in vivo ( Figure 3D,E,I,K). Nevertheless, inhibition of mTORC1 activity by rapamycin significantly prevented the neuritogenesis of RGCs ( Figure 3D-F), suggesting that the regenerative effects of HuMSC-exos were mTORC1 activitydependent.
In summary, our study shows that HuMSC-exos are a promising and viable regenerative therapy for ON injury that depends on specific miRNAs to regulate mTORC1 signalling. We also provide a better understanding of the important role of miR-222-3p and miR-22-3p delivered by HuMSC-exos in promoting axon regeneration. The schematic representations of our study are shown in Figure 4I and Figure S2H. Therefore, these results may provide new evidence for the role of HuMSC-exos and their exosomal miRNAs in axon regeneration after ON injury. Moreover, modified HuMSC-exos can be further transfected with appropriate miRNAs to augment their efficacy in neuroregenerative therapy.

A C K N O W L E D G E M E N T S
We acknowledge and appreciate all the colleagues who have participated in this study for their valuable efforts and comments on this paper. This work was supported by grants from the Natural Science Foundation of China (81961128021, 81870682), the National Key R&D Program of China (2022YEF0203200), the Postdoctoral Research Foundation of China (No. 2020M683078), the Guangdong Provincial Frontier and Key Technology Innovation special fund (2015B020227001).